Developing genetic tools for a new model of infection and immunity

描述(申请人提供)：寄生性线虫(PN)感染仍然是全球人类健康的主要威胁，有超过10亿人感染。儿童、孕妇和老年人特别容易受到线虫感染的影响。控制策略仅限于对感染者进行定期驱虫，这受到快速再感染率和抗药性蠕虫种群发展的限制。目前还没有针对人类PN感染的疫苗。新药和疫苗的开发将需要更好地了解PN生物学，特别是感染过程和宿主对感染的免疫反应。对专属宿主的要求和缺乏良好的动物模型限制了对PNS感染和篡夺宿主免疫反应的机制的研究。虽然自由生活的线虫秀丽线虫是线虫发展的极好模式，但它不是一种寄生虫。昆虫寄生线虫细菌节异杆线虫(Heterorhabditis bligiophora，HB)具有线虫的许多优点，包括易于培养和操纵所有生活史阶段，因此有可能成为一种易于处理的PN感染模型。然而，许多强大的遗传工具，如转基因和RNA干扰，在乙肝中还没有完全开发出来，限制了它作为广泛模型的使用。我们建议在乙肝中开发这些遗传工具，重点针对感染的幼虫和早期寄生虫阶段，以研究感染机制。我们将采取两种平行但独立的方法来开发这些工具，这将为未来对PN感染机制的研究提供基础。在目标1中，我们提出了一种通过种系显微注射基于piggyBac反转录转座子的整合载体来将报告基因导入到Hb染色体中的方法。在目标2中，我们将为第二代Hb两性人开发一个强大的RNAi协议。通过专注于第二代两性动物，我们处于理想的位置来探索PN感染的生物学，因为这一阶段的后代是感染阶段。这种新型线虫模型中基因敲除(反向遗传学)和转基因方法的发展将代表着PN研究的重大进展，并将使首次研究PN基因在感染过程中的功能成为可能。

DESCRIPTION (provided by applicant): Parasitic nematode (PN) infections remain a major threat to human health worldwide, with more than 1 billion people infected. Children, pregnant women, and the elderly are particularly susceptible to morbidity from nematode infection. Control strategies are restricted to periodic de-worming of infected individuals, which is limited by rapid re-infection rates and the development of drug resistant worm populations. There are no vaccines available for PN infections in humans. Development of new drugs and vaccines will require a better understanding of PN biology, particularly the infective process and the host's immune response to infection. The requirement of an obligate host and the lack of good animal models have limited investigations into mechanisms by which PNs infect and usurp the host immune response. While the free living nematode Caenorhabditis elegans is an excellent model for nematode development, it is not a parasite. The insect parasitic nematode Heterorhabditis bacteriophora (Hb) offers potential as a tractable model of PN infection that has many of the advantages of C. elegans, including easy culture and manipulation of all life history stages. However, many of the powerful genetic tools, like transgenesis and RNA interference, have not been fully developed in Hb, restricting its use as a widespread model. We propose to develop these genetic tools in Hb with a focus on targeting the infective juvenile and early parasitic stages in order to investigate infection mechanisms. We will take two parallel but independent approaches to develop these tools that will provide a foundation for future investigation into the mechanism of PN infection. In Aim 1, we propose to develop a transfection protocol for Hb via germ-line microinjection of a piggybac retrotransposon-based integrating vector to introduce the reporter transgenes into Hb chromosomes. In Aim 2, we will develop a robust RNAi protocol for 2nd generation Hb hermaphrodites. By focusing on 2nd generation hermaphrodites we are ideally positioned to probe the biology of PN infection, as the progeny of this stage are the infective stage. Development of methods for gene knock down (reverse genetics) and transfection in this novel model nematode would represent a significant advance for PN research, and will allow investigation of PN gene function during infection for the first time.